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The treatment of chronic wounds still challenges modern medicine because of these wounds' heterogenic pathophysiology. Processes such as inflammation, ischemia and bacterial infection play major roles in the progression of a chronic wound. In recent years, preclinical wound models have been used to understand the underlying processes of chronic wound formation. However, the wound models used to investigate chronic wounds often lack translatability from preclinical models to patients, and often do not take exaggerated inflammation into consideration. Therefore, we aimed to investigate prolonged inflammation in a porcine wound model by using resiquimod, a TLR7 and TLR8 agonist. Pigs received full thickness excisional wounds, where resiquimod was applied daily for 6 days, and untreated wounds served as controls. Dressing change, visual documentation and wound scoring were performed daily. Biopsies were collected for histological as well as gene expression analysis. Resiquimod application on full thickness wounds induced a visible inflammation of wounds, resulting in delayed wound healing compared to non-treated control wounds. Gene expression analysis revealed high levels of IL6, MMP1 and CD68 expression after resiquimod application, and histological analysis showed increased immune cell infiltration. By using resiquimod, we were able to show that prolonged inflammation delayed wound healing, which is often observed in chronic wounds in patients. The model we used shows the importance of inflammation in wound healing and gives an insight into the progression of chronic wounds.

In burn injuries, risk factors and limitations to treatment success are difficult to assess clinically. However, local cellular responses are characterized by specific gene-expression patterns. MicroRNAs (miRNAs) are single-stranded, non-coding RNAs that regulate mRNA expression on a posttranscriptional level. Secreted through exosome-like vesicles (ELV), miRNAs are intracellular signalers and epigenetic regulators. To date, their role in the regulation of the early burn response remains unclear. Here, we identified 43 miRNAs as potential regulators of the early burn response through the bioinformatics analysis of an existing dataset. We used an established human ex vivo skin model of a deep partial-thickness burn to characterize ELVs and miRNAs in dermal interstitial fluid (dISF). Moreover, we identified miR-497-5p as stably downregulated in tissue and dISF in the early phase after a burn injury. MiR-218-5p and miR-212-3p were downregulated in dISF, but not in tissue. Target genes of the miRNAs were mainly upregulated in tissue post-burn. The altered levels of miRNAs in dISF of thermally injured skin mark them as new biomarker candidates for burn injuries. To our knowledge, this is the first study to report miRNAs altered in the dISF in the early phase of deep partial-thickness burns.

Traditionally, cutaneous drug delivery is studied by skin accumulation or skin permeation, while alternative techniques may enable the interactions between the drug and the skin to be studied in more detail. Time-resolved skin profiling for pharmacokinetic monitoring of two Janus Kinase (JAK) inhibitors, tofacitinib and LEO 37319A, was performed using dermal open-flow microperfusion (dOFM) for sampling of perfusate in an ex vivo and in vivo setup in pig skin. Additionally, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed to investigate depth-resolved skin distributions at defined time points ex vivo in human skin. By dOFM, higher skin concentrations were observed for tofacitinib compared to LEO 37319A, which was supported by the lower molecular weight, higher solubility, lipophilicity, and degree of protein binding. Using MALDI-MSI, the two compounds were observed to show different skin distributions, which was interpreted to be caused by the difference in the ability of the two molecules to interact with the skin compartments. In conclusion, the techniques assessed time- and depth-resolved skin concentrations and were able to show differences in the pharmacokinetic profiles of two JAK inhibitors. Thus, evidence shows that the two techniques can be used as complementary methods to support decision making in drug development.

Objective: To implement OFM-recirculation and OFM-suction capable of direct and absolute in-vivo quantification of albumin in the ISF of pigs., Approach: OFM-recirculation and OFM-suction were used to collect ISF in-vivo in pigs and lymph was collected from the same pigs after OFM sampling. Blood was collected before and after OFM sampling, plasma was isolated and mean albumin plasma concentrations per pig were used to yield albumin ISF-to-plasma ratios. We characterized the quality of the collected undiluted ISF via (1) stable albumin ISF-to-plasma ratio in OFM-recirculation and in OFM-suction samples, (2) comparison of albumin ISF-to-plasma ratios from OFM-recirculation and OFM-suction and (3) comparison of normalized albumin concentrations in the ISF and lymph., Main Results: Both advanced OFM methods were successfully implemented and albumin was quantified from the collected ISF samples. OFM-recirculation reached stable albumin ISF-to-plasma ratios after 20 recirculation cycles. Absolute ISF albumin concentrations were 11.2 mg ml -1 (OFM-recirculation) and 14.2 mg ml -1 (OFM-suction). Albumin ISF-to-plasma ratios were 0.39 ± 0.04 (OFM -recirculation) and 0.47 ± 0.1 (OFM-suction)., Significance: Knowledge of the ISF protein content is of major importance when assessing PK/PD effects, especially of highly protein bound drugs. Up to now, only blood albumin values have been available to determine the degree of protein binding in several tissues. OFM-recirculation and OFM-suction allow direct, absolute quantification of albumin in ISF for the first time and enable investigation of the degree of protein binding of a drug directly in its target tissue.

Burn injuries initiate numerous processes such as heat shock response, inflammation and tissue regeneration. Reliable burn models are needed to elucidate the exact sequence of local events to be able to better predict when local inflammation triggers systemic inflammatory processes. In contrast to other ex vivo skin culture approaches, we used fresh abdominal skin explants to introduce contact burn injuries. Histological and ultrastructural analyses confirmed a partial-thickness burn pathology. Gene expression patterns and cytokine production profiles of key mediators of the local inflammation, heat shock response, and tissue regeneration were analyzed for 24 h after burn injury. We found significantly increased expression of factors involved in tissue regeneration and inflammation soon after burn injury. To investigate purely inflammation-mediated reactions we injected lipopolysaccharide into the dermis. In comparison to burn injury, lipopolysaccharide injection initiated an inflammatory response while expression patterns of heat shock and tissue regeneration genes were unaffected for the duration of the experiment. This novel ex vivo human skin model is suitable to study the local, early responses to skin injuries such as burns while maintaining an intact overall tissue structure and it gives valuable insights into local mechanisms at the very beginning of the wound healing process after burn injuries.

Background: Burn wound progression is a significant problem as burns initially thought to be superficial can actually become full thickness over time. Cooling is an efficient method to reduce burn wound conversion. However, if the cooling agent is below room temperature, depending on the wound size the patient is at risk of hypothermia. Additionally, tissue perfusion is reduced leading to an aggravation of burn wound progression. We investigated if wound dressings based on non-pre-cooled bacterial nanocellulose (BNC) with a high water content cool a burn just by evaporation and reduce the intradermal damages in the skin., Material and Methods: In a human ex-vivo model, skin explants underwent contact burns using a 100 °C hot steel block. The burned areas were divided into two groups of which one was cooled with a BNC-based wound dressing. Intradermal temperature probes measured temperature in cooled and uncooled burn sites over 24 h. For histological assessments of the burned areas biopsies were taken at different time points. High mobility group box-1 (HMBG1) staining served as marker for cell vitality and necrosis in the different skin layers., Results: Intradermal temperature measurement showed that application of the BNC-based wound dressing reduced temperature significantly in burned skin. This cooling effect resulted in a maximum temperature difference of 6.4 ± 1.9 °C and a significant mean reduction of the area under the curve in the first hour after burn of 62% (p < 0.0001). The histological results showed less necrosis and less dermal-epidermal separation in the cooled areas. The HMGB1 staining revealed more vital cells in the cooled group than in the uncooled group., Conclusion: Based on our results, BNC-based wound dressings cool a burn. Intradermal temperature as well as thermal damage of the tissue was reduced. The tested BNC-based wound dressing can be used without pre-cooling to cool a burn as well as to reduce the burn BNC-based wound progression through its evaporation cooling effect., (Copyright © 2020 Elsevier Ltd and ISBI. All rights reserved.)

Purpose: To investigate the difference in clinical efficacy in AD patients between two topical PDE4 inhibitors using dermal open flow microperfusion and cAMP as a pharmacodynamic read-out in fresh human skin explants., Methods: Clinical formulations were applied to intact or barrier disrupted human skin explants and both skin biopsy samples and dermal interstitial fluid was sampled for measuring drug concentration. Furthermore, cAMP levels were determined in the skin biopsies as a measure of target engagement., Results: Elevated cAMP levels were observed with LEO 29102 while no evidence of target engagement was obtained with LEO 39652. In barrier impaired skin the dISF concentration of LEO 29102 was 2100 nM while only 33 nM for LEO 39652. For both compounds the concentrations measured in skin punch biopsies were 7-33-fold higher than the dISF concentrations., Conclusions: Low unbound drug concentration in dISF in combination with minimal target engagement of LEO 39652 in barrier impaired human skin explants supports that lack of clinical efficacy of LEO 39652 in AD patients is likely due to insufficient drug availability at the target. We conclude that dOFM together with a pharmacodynamic target engagement biomarker are strong techniques for establishing skin PK/PD relations and that skin biopsies should be used with caution.

Purpose: Dermal open flow microperfusion (dOFM) has previously demonstrated its utility to assess the bioequivalence (BE) of topical drug products in a clinical study. We aimed to characterize the sources of variability in the dermal pharmacokinetic data from that study., Methods: Exploratory statistical analyses were performed with multivariate data from a clinical dOFM-study in 20 healthy adults evaluating the BE, or lack thereof, of Austrian test (T) and U.S. reference (R) acyclovir cream, 5% products., Results: The overall variability of logAUC values (CV: 39% for R and 45% for T) was dominated by inter-subject variability (R: 82%, T: 91%) which correlated best with the subject's skin conductance. Intra-subject variability was 18% (R) and 9% (T) of the overall variability; skin treatment sites or methodological factors did not significantly contribute to that variability., Conclusions: Inter-subject variability was the major component of overall variability for acyclovir, and treatment site location did not significantly influence intra-subject variability. These results support a dOFM BE study design with T and R products assessed simultaneously on the same subject, where T and R treatment sites do not necessarily need to be next to each other. Localized variation in skin microstructure may be primarily responsible for intra-subject variability.

Background: Bacterial nanocellulose (BNC) is considered a promising carrier for various substances and novel approaches using BNC in combination with antiseptics are well documented. However, the difference in the molecular weight of these molecules influences their uptake by and release from BNC. Analysing the diffusion of standard molecules with different weight, e.g. dextrans, offers the possibility to investigate the mobility of various molecules. We aimed to test the use of BNC regarding uptake and release of different standard molecules as well as two commercially available antiseptics for possible applications in future wound dressings., Material and Methods: Diffusion profiles, uptake and release of three FITC-dextran molecules differing in weight as well as octenidine (Octenisept®) and povidone-iodine (Betaisodona®)-based antiseptics were tested with BNC-based wound dressings. Furthermore, the antiseptic efficacy of BNC in combination with antiseptics against Staphylococcus aureus was tested., Results: Uptake and release capacity for FITC-dextran molecules showed a molecular weight-dependent mobility from BNC into an agarose gel. The loading capacity of BNC was also inversely proportional to the molecular weight of the antiseptics. The release test for octenidine showed a sustained and prolonged delivery into a solid matrix, whereas povidone-iodine was released faster. Both antiseptic solutions combined with BNC showed a good dose-dependent efficacy against S. aureus., Conclusion: Results obtained from the mobility of FITC-dextran molecules in the BNC matrix could open possible applications for the combination of BNC with other molecules for medical applications. Combination of both tested antiseptics with BNC showed to be an efficient approach to control bacterial infections., (Copyright © 2019 Elsevier Ltd and ISBI. All rights reserved.)

Continuous subcutaneous insulin infusion (CSII) is a widely used treatment for diabetes patients. Insulin infusion sets (CSII-catheters) are continuously optimized regarding size, handling and safety, but recurring dysfunction (kinking or occlusion), due to different user situations, behavior or chain of events, demand new ways to improve the functionality and safety in patients experiencing these issues. A novel CSII-catheter design (Lantern) features additional lateral perforations, which guarantee functionality even in case of kinking or occlusion. This study aimed to compare functionality, insulin distribution, and failure rate of Lantern and standard catheters using excised human adipose tissue samples. Novel Lantern CSII-catheters (open and artificially occluded) and commercially available standard CSII-catheters were inserted into adipose tissue samples. A mixture of insulin and contrast agent was infused as single bolus (7 IU) with an insulin infusion pump at highest flow rate (1 IU/s). Microtomography images and surface-to-volume ratios were used to assess insulin distribution and depot volume indicating the functionality of CSII-catheters. Failure rate was measured by flow-stop alerts of the pump. We found no difference in the volume of insulin depots compared with the nominal volume of 70 μL. Surface-to-volume ratios showed no significant difference among CSII-catheters. None of the catheters triggered any flow-stop alarm. The novel Lantern CSII-catheter design achieved similar insulin distribution as commercially available CSII-catheters. Moreover, functionality of Lantern CSII-catheters was guaranteed during occlusion, which is an improvement compared with standard CSII-catheters. We conclude that the novel CSII-catheter design has the potential to provide a valuable contribution to patient well-being and safety.

Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Drugs for neurological diseases have to cross the blood-brain barrier (BBB) to induce their therapeutic effect. In vivo drug quantification in the brain is challenging, because invasive methods damage the BBB and measurement results may be confounded by drug leakage from the blood into the brain through the disrupted BBB. Cerebral open flow microperfusion (cOFM) is an in vivo sampling technique that allows BBB healing and re-establishment after probe implantation and before sampling is performed. It therefore provides the opportunity to sample compounds in cerebral interstitial fluid with an intact BBB. This article comprehensively describes the experimental setup and procedures, perfusate requirements, critical parameters, common problems that may occur, and their causes and solutions. Typical results from a cOFM sampling experiment are presented and discussed. This protocol provides a tool for performing pharmacokinetic and pharmacodynamic studies in mouse or rat brain with an intact BBB. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

When people judge the duration of stimuli, judgments are influenced by the physical size of these stimuli. Specifically, people tend to judge the duration of large stimuli longer than the duration of small stimuli. However, some authors (Bottini & Casasanto, 2010; Ma, Yang, & Zhang, 2012) have reported that even implicit size can affect duration estimates. The present study extends the results of these studies. Specifically, we examined whether the imagined size of objects would also influence duration estimates. In each trial, participants reproduced the duration of an animal word. During the presentation of the word, they were asked to imagine the word's referent. The animals employed in all experiments varied in size. In Experiment 1, participants were instructed to imagine the animals in a fixed context. Reproduced duration increased not only with the numbers of letters of the animal word (i.e., physical size) but also with the size of the word's referent (i.e., implicit size of the animal). Experiments 2 and 3 examined whether the effect of size would increase when the size of the animals was made more salient. The results of all experiments showed that reproduced duration increases with implicit stimulus size. In addition, Experiments 2 and 3 provide some evidence that the imagined size effect becomes more pronounced when the participant's attention is drawn to the size differences among the imagined animals. It is argued that the implicit size effect is mediated by mental imagery that operates on pre-existing space-time associations. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Background: With the increase of antimicrobial resistance in recent decades, other methods of preventing and fighting infections must be considered. Burn patients, whose wound areas are often extensive, are especially prone to wound infections. The loading of bacterial nanocellulose (BNC) with antiseptics has already been successfully performed but unfortunately, the described procedure is time-consuming and thus not applicable in a clinical emergency setting. Therefore, a clinically feasible approach was established., Material and Methods: Sheets of BNC-based wound dressings were placed into antiseptic solutions containing PHMB (Prontosan ® and LAVANID ® 2) and were left to soak for up to two hours. At different time points, samples were analysed for their concentration of PHMB and antiseptic efficacy., Results: Within 30min, clinically relevant concentrations of PHMB were achieved in the BNC-based wound dressing. The 30-min PHMB uptake for Prontosan ® and LAVANID ® 2 resulted in concentrations of 0.05% and 0.019%, respectively. Samples from the PHMB loaded dressing showed a dose dependent antiseptic efficacy for Staphylococcus aureus., Conclusion: This experiment showed that the loading of BNC-based wound dressings with PHMB-containing antiseptics was achieved by a simple and quick procedure. According to studies a PHMB concentration of 0.001% can already inhibits all bacterial growth, indicating that the concentrations of PHMB in the BNC-based wound dressings after 30min are higher than the minimal inhibitory concentration and the antiseptic efficacy after 120min loading analysed by an standardized bacterial disk diffusion assay was shown to be comparable to the clinically used Suprasorb ® X+PHMB wound dressing., (Copyright © 2018. Published by Elsevier Ltd.)

Eicosanoids are lipid-mediator molecules with key roles in inflammatory skin diseases, such as psoriasis. Eicosanoids are released close to the source of inflammation, where they elicit local pleiotropic effects and dysregulations. Monitoring inflammatory mediators directly in skin lesions could provide new insights and therapeutic possibilities. Here, we analyzed dermal interstitial fluid samples obtained by dermal open-flow microperfusion in a rat model of skin inflammation. We developed a solid-phase extraction ultra-HPLC/MS/MS method to reliably and precisely analyze small-volume samples and quantified 11 eicosanoids [thromboxane B 2 , prostaglandin (PG) E 2 , PGD 2 , PGF 2α , leukotriene B 4 , 15-HETE, 12-HETE, 5-HETE, 12-hydroxyeicosapentaenoic acid, 13-HODE, and 17-hydroxydocosahexaenoic acid]. Our method achieved a median intraday precision of approximately 5% and interday precision of approximately 8%. All calibration curves showed excellent linearity between 0.01 and 50 ng/ml ( R 2 > 0.980). In the rat model, eicosanoids were significantly increased in imiquimod-treated inflamed skin sites compared with untreated control sites. Oral treatment with an anti-inflammatory glucocorticoid decreased eicosanoid concentrations. These results show that a combination of tissue-specific sampling with LC/MS analytics is well suited for analyzing small sample volumes from minimally invasive sampling methods such as open-flow microperfusion or microdialysis to study local inflammation and the effect of treatments in skin diseases., (Copyright © 2019 Pipper et al.)

Objective: Ischaemia is one of the biggest problems in wound healing. It causes chronic wounds and also prevents normal wound healing because the tissue is oxygen deprived. Most oxygen-supplying therapies are only feasible in a clinical setting, but topical haemoglobin applications, such as Granulox, can be used in a non-clinical setting. For home application, the haemoglobin solution is sprayed topically onto the wound using a pressurised ready-to-use device with a bag-on-valve system. Although this system does not mix product and propellant, the risk of product inhalation by the patient, user or bystanders has to be minimised. This safety study aimed to determine particle size and product concentration in the surroundings after application to determine if there is a risk that product particles enter the respiratory tract., Methods: Measurements were performed using a laser scattered light photometer and a Scanning Mobility Particle Sizer (SMPS)-Spectrometer at different distances from the measuring devices to determine the inhalation risk for a possible user, patient and bystander. At all measuring points the amount of particles, their size and the formation of dust were measured., Results: No nanoparticles or dust were created during the application of the haemoglobin spray. The concentrations of the measured particles are below the allowed limits defined by Austrian law., Conclusion: There is no risk of inhaling nanoparticles or being exposed to harmful concentrations of larger particles of the tested product. All the product's ingredients can be degraded and excreted by the human body through natural pathways.

Background: Assessment of drug concentration in the brain interstitial fluid (ISF) is crucial for development of brain active drugs, which are mainly small, lipophilic substances able to cross the blood-brain barrier (BBB). We aimed to compare the applicability of cerebral Open Flow Microperfusion (cOFM) and Microdialysis (MD) to sample the lipophilic substance amitriptyline (AMI), its metabolites Hydroxyamitriptyline (HYA), Nortriptyline (NOR), Amitriptyline-N-Oxide (ANO), deuterated water (D 2 O) and the hydrophilic substance sodium fluorescein (Naf) in brain ISF. NEW METHOD: cOFM has been refined to yield increased spatial resolution and performance., Comparison of Cofm and Md and Results: Performance of cOFM and MD was assessed by in vivo AUC ratios of probe samples (AUC COFM /AUC MD ) and the in vivo relative recovery of D 2 O (RR vv,D2O ). Adsorption of AMI and Naf to MD and cOFM was assessed by the in vitro relative recovery (RR vt ) prior to the in vivo experiments. The in vivo AUC ratio of AMI and RR vv,D2O was about two times higher for cOFM than for MD (AUC OFM /AUC MD  = 2.0, RR vv,D2O (cOFM)/RR vv,D2O (MD) = 2.1). cOFM detected all investigated AMI metabolites except NOR. MD did not detect HYA, NOR, ANO and Naf. In vitro adsorption of AMI and Naf to the MD membrane was strong (RR vt,AMI  = 4.4%, RR vt,Naf  = 1.5%) but unspecific adsorption to cOFM was negligibly small (RR vt,AMI  = 98% and RR vt,Naf  = 98%)., Conclusions: cOFM showed better performance when sampling AMI and its metabolites, Naf and D 2 O, and had an about two times higher RR vv,D2O than MD. MD did not detect HYA, NOR, ANO and Naf, most likely due to membrane adsorption., (Copyright © 2018. Published by Elsevier B.V.)

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7 f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7 f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7 f/f Tyr-Cre mice relative to Atg7 f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7 f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7 f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.

Objective: The inability of leptin to suppress food intake in diet-induced obesity, sometimes referred to as leptin resistance, is associated with several distinct pathological hallmarks. One prevailing theory is that impaired transport of leptin across the blood-brain barrier (BBB) represents a molecular mechanism that triggers this phenomenon. Recent evidence, however, has challenged this notion, suggesting that leptin BBB transport is acquired during leptin resistance., Methods: To resolve this debate, we utilized a novel cerebral Open Flow Microperfusion (cOFM) method to examine leptin BBB transport in male C57BL/6J mice, fed a chow diet or high fat diet (HFD) for 20 days., Results: Basal plasma leptin levels were 3.8-fold higher in HFD-fed mice (p < 0.05). Leptin administration (2.5 mg/kg) elicited similar pharmacokinetic profiles of circulating leptin. However, while leptin reduced food intake by 20% over 22 h in chow-fed mice, it did not affect food intake in HFD-fed mice. In spite of this striking functional difference, hypothalamic leptin levels, as measured by cOFM, did not differ between chow-fed mice and HFD-fed mice following leptin administration., Conclusions: These data suggest that leptin transport across the BBB is not impaired in non-obese leptin resistant mice and thus unlikely to play a direct role in the progression of pharmacological leptin resistance., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Previous studies have suggested that unexpected stimuli are perceived as being longer than expected ones (e.g., the temporal oddball effect). These studies manipulated stimulus expectation mostly via stimulus repetitions and stimulus probabilities. However, these manipulations might affect duration judgments not only through the modulation of stimulus expectation. Therefore, the present study introduces a novel paradigm to isolate the effect of stimulus expectation on perceived duration from repetition and probability effects. In 2 experiments, participants vocalized which of 2 possible stimuli they expected in each trial immediately before stimulus presentation (self-generated expectations). Following stimulus presentation, participants performed a temporal bisection task on the duration of the presented stimuli. For both color (Experiment 1) and shape stimuli (Experiment 2), longer perceived durations were observed when stimulus expectations were fulfilled rather than violated. These results contrast with previous studies from which it has been concluded that stimulus expectation shortens perceived duration. Instead, the findings are rather in line with the idea that higher level stimulus expectation enhances stimulus processing and thus prolongs subjective duration (Matthews & Gheorghiu, 2016). Importantly, this also challenges the assumption that higher level stimulus expectation is a key mechanism driving the temporal oddball effect. (PsycINFO Database Record, ((c) 2018 APA, all rights reserved).)

Previous studies have demonstrated that filled intervals are perceived as being longer than empty intervals of the same duration (i.e., the filled - duration illusion). In the present study, we asked whether the motor actions involved in filled and empty reproductions (a single continuous key press vs. two discrete key presses) would affect time estimates in a similar way. We hypothesized that reproductions of intervals of the same duration should be shorter for filled than empty reproductions (i.e., a filled - reproduction illusion). In two experiments, participants reproduced filled and empty intervals (ranging from 400 to 1,600 ms) using filled and empty reproductions. The results provided evidence for both kinds of illusions, even though the evidence was clearer for the filled - duration than for the filled - reproduction illusion. The present study demonstrates that in a situation in which both illusions work in concert, reproductions of the same interval can vary dramatically depending on the combination of interval and reproduction type.

Background: Restoration of the physiologic hepatic-to-peripheral insulin gradient may be achieved by either portal vein administration or altering insulin structure to increase hepatic specificity or restrict peripheral access. Basal insulin peglispro (BIL) is a novel, PEGylated basal insulin with a flat pharmacokinetic and glucodynamic profile and altered hepatic-to-peripheral action gradient. We hypothesized reduced BIL exposure in peripheral tissues explains the latter, and in this study assessed the adipose tissue interstitial fluid (ISF) concentrations of BIL compared with human insulin (HI)., Methods: A euglycemic glucose clamp was performed in patients with type 1 diabetes during continuous intravenous (IV) infusion of BIL or HI, while the adipose ISF insulin concentrations were determined using open-flow microperfusion (OFM). The ratio of adipose ISF-to-serum concentrations and the absolute steady-state adipose ISF concentrations were assessed using a dynamic no-net-flux technique with subsequent regression analysis., Results: Steady-state BIL concentrations in adipose tissue ISF were achieved by ∼16 h after IV infusion. Median time to reach steady-state glucose infusion rate across doses ranged between 8 and 22 h. The average serum concentrations (coefficient of variation %) of BIL and HI were 11,200 pmol/L (23%) and 425 pmol/L (15%), respectively. The ISF-to-serum concentration ratios were 10.2% for BIL and 22.9% for HI., Conclusions: This study indicates feasibility of OFM to measure BIL in ISF. The observed low ISF-to-serum concentration ratio of BIL is consistent with its previously demonstrated reduced peripheral action.

Background: The availability of generic topical dermatological drug products is constrained by the limited methods established to assess topical bioequivalence (BE). A novel cutaneous pharmacokinetic approach, dermal open-flow microperfusion (dOFM), can continuously assess the rate and extent to which a topical drug becomes available in the dermis, to compare in vivo dermal bioavailability (BA) and support BE evaluations for topical products., Objective: To evaluate whether dOFM is an accurate, sensitive, and reproducible in vivo method to characterize the intradermal BA of acyclovir from 5 % acyclovir creams, comparing a reference (R) product either to itself or to a different test (T) product., Methods: In a single-center clinical study, R or T products were applied to six randomized treatment sites on the skin of 20 healthy human subjects. Two dOFM probes were inserted in each treatment site to monitor the intradermal acyclovir concentration for 36 h. Comparative BA (of R vs. R and T vs. R) was evaluated based on conventional BE criteria for pharmacokinetic endpoints (area under the curve and maximum dermal concentration) where the 90 % confidence interval of the geometric mean ratio between the T and R falls within 0.80-1.25., Results: The positive control products (R vs. R) were accurately and reproducibly confirmed to be bioequivalent, while the negative control products (T vs. R) were sensitively discriminated not to be bioequivalent., Conclusions: dOFM accurately, sensitively, and reproducibly characterized the dermal BA in a manner that can support BE evaluations for topical acyclovir 5 % creams in a study with n = 40 (20 subjects in this study)., Competing Interests: Compliance with Ethical Standards Funding Funding for this project was made possible, in part, by the FDA through research award FD004946. The views expressed in this publication do not reflect the official policies of the FDA, or the Department of Health and Human Services; nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government. Conflict of interest MB, KIT, RR, BT, TA, CD, TB, SK, DS, TRP, and FS are employees of Joanneum Research holding patents on OFM devices. SGR is an employee of US FDA. GS and IK declare that there are no conflicts of interest. Ethics, informed consent, research involving human participants The study was conducted with the full informed consent of all participating subjects, under the authority of the Ethical Committee of the Medical University of Graz, the Austrian health authority AGES, and the FDA’s Research Involving Human Subjects Committee (RIHSC) and was performed in accordance with Good Clinical Practice and the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The study has been registered in the European Clinical Trials Register (EudraCT No. 2013-005062-19) and at ClinicalTrials.gov (NCT02711267).

(2)H2O as nonradioactive, stable marker substance is commonly used in preclinical and clinical studies and the precise determination of (2)H2O concentration in biological samples is crucial. However, aside from isotope ratio mass spectrometry (IRMS), only a very limited number of methods to accurately measure the (2)H2O concentration in biological samples are routinely established until now. In this study, we present a straightforward method to accurately measure (2)H-enrichment of rat brain interstitial fluid (ISF) and rat plasma to determine the relative recovery of a cerebral open flow microperfusion (cOFM) probe, using headspace-gas-chromatography - quadrupole-mass-spectrometry. This method is based on basic-catalyzed hydrogen/deuterium exchange in acetone and detects the (2)H-labelled acetone directly by the headspace GC-MS. Small sample volumes and limited number of preparation steps make this method highly competitive. It has been fully validated. (2)H enriched to 8800 ppm in plasma showed an accuracy of 98.9% and %Relative Standard Deviation (RSD) of 3.1 with n = 18 over three days and with two operators. Similar performance was obtained for cerebral ISF enriched to 1100 ppm (accuracy: 96.5%, %RSD: 3.1). With this highly reproducible method we demonstrated the successful employment of (2)H2O as performance marker for a cOFM probe., (Copyright © 2016. Published by Elsevier Inc.)

Cerebral open flow microperfusion (cOFM) is a new in-vivo technique for continuous sampling of the interstitial fluid in brain tissue. cOFM can be used to monitor substance transport across the blood-brain barrier (pharmacokinetics) and to investigate metabolic changes in brain tissue after drug application (pharmacodynamics). The possibility of long-term implantation into the brain makes cOFM an outstanding tool in the development of brain relevant pharmaceutics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fiedler, Schröter, and Ulrich (2013) reported faster responses to a single written word when the semantic content of this word (e.g., "elephant") matched both targets (e.g., "animal", "gray") as compared to a single target (e.g., "animal", "brown"). This semantic redundancy gain was explained by statistical facilitation due to a race of independent memory retrieval processes. The present experiment addresses one alternative explanation, namely that semantic redundancy gain results from multiple pre-activation of words that match both targets. In different blocks of trials, participants performed a redundant-targets task and a lexical decision task. The targets of the redundant-targets task served as primes in the lexical decision task. Replicating the findings of Fiedler et al., a semantic redundancy gain was observed in the redundant-targets task. Crucially, however, there was no evidence of a multiple semantic priming effect in the lexical decision task. This result suggests that semantic redundancy gain cannot be explained by multiple pre-activation of words that match both targets., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Two experiments investigated the effects of stimulus repetition vs. stimulus novelty on perceived duration. In a reminder task, a standard and a comparison stimulus were presented consecutively in each trial, and the comparison was either a repetition of the standard or a different stimulus. Pseudowords (Experiment 1) or strings of consonants (Experiment 2) were used as stimuli and the inter-stimulus interval (ISI) between the standard and the comparison was either constant or variable. Participants were asked to judge whether the comparison was shorter or longer than the standard. In both experiments, we observed shorter judged durations for repeated than for novel comparisons whereas the manipulation of the ISI had no pronounced effects on duration judgments. The finding of shorter duration judgments for repeated as compared to novel nonwords replicates the results of a previous study (Matthews, 2011) which employed highly complex stimulus material. The present study shows that changes of simple, semantically meaningless stimuli are sufficient to result in a shorter perceived duration of repeated as compared to novel stimuli.

Three experiments assessed whether participants perceive infrequent stimuli (oddballs) to last longer than frequent ones (standards). The classical oddball paradigm was modified so that participants judged the duration of a target stimulus which could either be a standard or an oddball. The target was always the fifth stimulus in the stream and all stimuli were presented at predefined spatial positions. These modifications enabled a direct comparison of duration judgments for oddballs and standards. In Experiments 1 and 2 not only the duration of oddballs but also the duration of standards was overestimated by virtually the same amount. In other words, the overestimation of oddballs was not due to their oddness but reflected a different temporal dilation such as the negative time-order error. In Experiment 3, all stimuli were presented at the same spatial position. Again, both oddballs and standards were overestimated, however, oddballs more so. The present results highlight the importance of comparing the judged duration of oddballs and standards when evaluating the size of the genuine oddball effect. Furthermore, the findings suggest that the occurrence of temporal oddball effects can depend on spatial features of stimulus presentation.

An elaborated diffusion process model (a Diffusion Model for Conflict Tasks, DMC) is introduced that combines conceptual features of standard diffusion models with the notion of controlled and automatic processes. DMC can account for a variety of distributional properties of reaction time (RT) in conflict tasks (e.g., Eriksen flanker, Simon, Stroop). Specifically, DMC is compatible with all observed shapes of delta functions, including negative-going delta functions that are particularly challenging for the class of standard diffusion models. Basically, DMC assumes that the activations of controlled and automatic processes superimpose to trigger a response. Monte Carlo simulations demonstrate that the unfolding of automatic activation in time largely determines the shape of delta functions. Furthermore, the predictions of DMC are consistent with other phenomena observed in conflict tasks such as error rate patterns. In addition, DMC was successfully fitted to experimental data of the standard Eriksen flanker and the Simon task. Thus, the present paper reconciles the prominent and successful class of diffusion models with the empirical finding of negative-going delta functions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Temporal preparation usually results in enhanced performance in choice reaction time tasks. The present study investigated to what extent temporal preparation involves increased readiness for task-specific processing requirements as opposed to increased task-independent readiness. Participants performed either a pitch, a letter, or a color discrimination task within a variable foreperiod paradigm and tasks alternated regularly between auditory and visual discriminations. In separate blocks of trials, the upcoming visual discrimination task was either predictable or unpredictable. We observed the standard variable foreperiod effect for both visual discrimination tasks irrespective of task predictability. Importantly, however, the variable foreperiod effect was larger when the visual discrimination task was predictable than when it was unpredictable. These results suggest that temporal preparation in choice reaction time tasks involves increased readiness for both task-independent and task-specific processing requirements.

The neuroprotective blood-brain barrier (BBB) keeps many drug candidates below therapeutic levels in the central nervous system. Glutathione PEGylated liposomal doxorubicin (2B3-101) has been developed to safely enhance the delivery of doxorubicin to brain tumors. However, doxorubicin concentration in extracellular brain fluid cannot yet be reliably measured using conventional techniques. Cerebral open flow microperfusion (cOFM), a recently developed sampling technique, allows monitoring of drug concentrations in the brain independent of molecular weight and lipophilicity. In combination with cOFM sampling, sodium fluorescein (NaF) is used as a marker for BBB integrity. Rats received one intravenous dose of 7 mg/kg of either 2B3-101 or PEGylated liposomal doxorubicin (generic Caelyx(®)). Blood and cOFM sampling was performed for 5 h after dose injection. NaF concentration in the brain was monitored and remained low indicating an intact BBB. The brain-to-blood ratio of doxorubicin was 4.8-fold higher after administration of 2B3-101 as compared with generic Caelyx(®) (p = 0.0016). In conclusion, by using cOFM it was possible to show that 2B3-101 leads to enhanced doxorubicin concentration in the brain without affecting the BBB integrity., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Blood-brain barrier (BBB) impairment in systemic inflammation leads to neuroinflammation. Several factors including cytokines, chemokines and signal transduction molecules are implicated in BBB dysfunction in response to systemic inflammation. Here, we have adopted a novel in vivo technique; namely, cerebral open flow microperfusion (cOFM), to perform time-dependent cytokine analysis (TNF-alpha, IL-6 and IL-10) in the frontal cortex of the rat brain in response to a single peripheral administration of lipopolysaccharide (LPS). In parallel, we monitored BBB function using sodium fluorescein as low molecular weight reporter in the cOFM sample. In response to the systemic LPS administration, we observed a rapid increase of TNF-alpha in the serum and brain, which coincides with the BBB disruption. Brain IL-6 and IL-10 synthesis was delayed by approximately 1 h. Our data demonstrate that cOFM can be used to monitor changes in brain cytokine levels and BBB disruption in a rat sepsis model.

The duration of rare stimuli (oddballs) presented within a stream of homogenous standards tends to be overestimated. This temporal oddball effect (OE) has been attributed to perceptual processes. The OE is usually assessed with a comparative judgment task. It has been argued, however, that this task is prone to decision biases. The present experiments employed comparative and equality judgments, since it has been suggested that equality judgments are less vulnerable to such biases. Experiments 1a and 1b used visual stimuli, and Experiment 2 auditory stimuli. The results provide no strong evidence for decision biases influencing the OE. In addition, computational modeling clearly suggests that the equality judgment is not particularly suited to distinguish between perceptual and decisional effects. Taken together, the pattern of the present results is most consistent with a perceptual origin of the OE.

This study investigated the histological tissue reaction to long-term implanted cerebral open flow microperfusion (cOFM) probes in the frontal lobe of the rat brain. Most probe-based cerebral fluid sampling techniques are limited in application time due to the formation of a glial scar that hinders substance exchange between brain tissue and the probe. A glial scar not only functions as a diffusion barrier but also alters metabolism and signaling in extracellular brain fluid. cOFM is a recently developed probe-based technique to continuously sample extracellular brain fluid with an intact blood-brain barrier. After probe implantation, a 2 week healing period is needed for blood-brain barrier reestablishment. Therefore, cOFM probes need to stay in place and functional for at least 15 days after implantation to ensure functionality. Probe design and probe materials are optimized to evoke minimal tissue reaction even after a long implantation period. Qualitative and quantitative histological tissue analysis revealed no continuous glial scar formation around the cOFM probe 30 days after implantation and only a minor tissue reaction regardless of perfusion of the probe.

The blood-brain barrier (BBB) limits substance transport to the brain and is therefore the major hurdle to overcome when developing neuroactive drugs. Herein, we report on cerebral open flow microperfusion (cOFM) as a new membrane-free technique for measuring substance transport across the intact BBB. The cOFM technique is based on a probe that is inserted into the brain, rupturing the BBB. The BBB is re-established within 15 days, which then allows sampling of interstitial brain fluid under physiological conditions. The aims of the present proof-of-concept study were to: (i) determine the time between cOFM probe insertion and BBB re-establishment; and (ii) demonstrate the ability of cOFM to sample the interstitial cerebral fluid with an intact BBB. The cOFM probe was inserted into the frontal lobe of Sprague-Dawley rats, resulting in BBB rupture. Re-establishment of the BBB was determined using Evans blue (EB) dye, which is an established marker for BBB intactness because it does not cross the intact BBB. Evaluating EB levels in the brain tissue indicated that the BBB was healed 11 days after probe insertion. To demonstrate transport across the healed BBB, we used sodium fluorescein (Naf), a sensitive, low molecular weight marker that can cross the intact BBB and can be used to monitor changes in BBB permeability. Significantly increased Naf levels were found in the interstitial fluid when hyperosmolar mannitol (known to open the BBB) was introduced via cOFM, which indicated partial opening of the BBB surrounding the cOFM probe. In conclusion, we show herein that cOFM allows monitoring of BBB permeability, which should be useful for measuring pharmacokinetics across the BBB and pharmacodynamics in the brain., (© 2013 Wiley Publishing Asia Pty Ltd.)

Objective: We investigated the impact of two different injection strategies on the pharmacokinetics and pharmacodynamics of insulin aspart in vivo in an open-label, two-period crossover study and verified changes in the surface-to-volume ratio ex vivo., Research Design and Methods: Before the clinical trial, insulin aspart was injected ex vivo into explanted human abdominal skin flaps. The surface-to-volume ratio of the subcutaneous insulin depot was assessed by microfocus computed tomography that compared 1 bolus of 18 IU with 9 dispersed boluses of 2 IU. These two injection strategies were then tested in vivo, in 12 C-peptide-negative type 1 diabetic patients in a euglycemic glucose clamp (glucose target 5.5 ± 1.1 mmol/L) for 8 h after the first insulin administration., Results: The ex vivo experiment showed a 1.8-fold higher mean surface-to-volume ratio for the dispersed injection strategy. The maximum glucose infusion rates (GIR) were similar for the two strategies (10 ± 4 vs. 9 ± 4; P = 0.5); however, times to reach maximum GIR and 50% and 10% of the maximum GIR were significantly reduced by using the 9 × 2 IU strategy (68 ± 33 vs. 127 ± 93 min; P = 0.01; 38 ± 9 vs. 49 ± 16 min; P < 0.01; 23 ± 6 vs. 30 ± 10 min; P < 0.05). For 9 × 2 IU, the area under the GIR curve was greater during the first 60 min (219 ± 89 vs. 137 ± 75; P < 0.01) and halved until maximum GIR (242 ± 183 vs. 501 ± 396; P < 0.01); however, it was similar across the whole study period (1,361 ± 469 vs. 1,565 ± 527; P = 0.08)., Conclusions: A dispersed insulin injection strategy enhanced the effect of a fast-acting insulin analog. The increased surface-to-volume ratio of the subcutaneous insulin depot can facilitate insulin absorption into the vascular system.

Previous studies provided evidence of the claim that the prediction of occluded action involves real-time simulation. We report two experiments that aimed to study how real-time simulation is affected by simultaneous action execution under conditions of full, partial or no overlap between observed and executed actions. This overlap was analysed by comparing the body sides and the movement kinematics involved in the observed and the executed action. While performing actions, participants observed point-light (PL) actions that were interrupted by an occluder, followed by a test pose. The task was to judge whether the test pose depicted a continuation of the occluded action in the same depth angle. Using a paradigm proposed by Graf et al., we independently manipulated the duration of the occluder and the temporal advance of the test pose relative to occlusion onset (occluder time and pose time, respectively). This paradigm allows the assessment of real-time simulation, based on prediction performance across different occluder time/pose time combinations (i.e., improved task performance with decreasing time distance between occluder time and pose time is taken to reflect real-time simulation). The PL actor could be perceived as from the front or back, as indicated by task instructions. In Experiment 1 (front view instructions), evidence of action simulation was obtained for partial overlap (i.e., observed and performed action corresponded either in body side or movement kinematics), but not for full or no overlap conditions. The same pattern was obtained in Experiment 2 (back view instructions), ruling out a spatial compatibility explanation for the real-time pattern observed. Our results suggest that motor processes affect action prediction and real-time simulation. The strength of their impact varies as a function of the overlap between observed and executed actions., (Copyright © 2011 Elsevier Inc. All rights reserved.)